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Nuclear Copyright

Medicine & Biology, Vol. 0 1997 Elsevier Science

24, 171-178, Inc.



0969-8051/97/$17.00 + 0.00 PI1 SO969-8051(96)00211-9


Fluorine4 8-Labeled [Nle4,D-Phe7]-a-MSH, an cx-Melanocyte Stimulating Hormoa Analogue

Gunesan Vaidyanathan and Michael R. Zalutsky

The cx-melanocyte stimulating hormone (wMSH) analogue [Nle4,D-Phe7]-cx-MSH was lain >80% radiochemical yield. The with *F using N-succinimidyl 4-[ *F]fluorobenzoate ([ FJSFB) values of [Nle4,D-Phe7]-cw-MSH and para-fluorobenzoyl-[Nle4,n-Phe7]-cx-MSH ([Nle4,D-Phe7,LysIGO ([Nle4,D(sF)PFB]-~-MSH) for inhibiting the binding of meta-[ 1311]iodobenzoyl-[N1e4,D-Phe7]-cx-MSH murine melanoma cells were 89 2 9 pM and 112 * 22 pM, Phe7,Lys1-(311)MIB]-cx-MSH) to B16-Fl respectively, suggesting that addition of 4-fluorobenzoate did not compromise cx-MSH receptor binding was influenced by the specific activity of the affinity. Binding of [Nle4,D-Phe7,Lys l-(lsF)PFB]-~-MSH preparation (400-1000 Ci/mmol). The normal tissue clearance of [Nle4,D-Phe7,Lyse( 18F)PFB]-~-MSH in NUCL MED BIOL 24;2:171-178, 1997. @ 1997 mice was quite rapid, with little evidence for defluorination. Elsevier Science Inc. beled








The incidence of malignant melanoma is increasing rapidly, making the development of improved methods for the detection and staging of this disease of paramount importance (18). This has motivated the development of monoclonal antibodies (MAbs) reactive with human melanoma-associated antigens that have been labeled with a variety of radionuclides and investigated as imaging agents (6). Unfortunately, the clinical impact of radioimmunoscintigraphy in patients with melanoma has been less than desired. Because the superior imaging characteristics of PET might enhance tumor detectability, we investigated the feasibility of labeling the F(ab), fragment of Mel-14, an antibody reactive with the chondroitin sulfate proteoglycan present in melanomas (7), with sF (14, 31). Although specific in vitro tumor binding and murine xenograft uptake with F-labeled Mel-14 F(ab), could be achieved, subsequent studies in dogs suggested that the clearance of activity from normal tissues was too slow to permit tumor imaging within a time frame compatible with the half-life of F (20). Another approach for the targeting of radionuclides, which should be more suitable for use with short half-life radionuclides, is the utilization of biologically active peptides reactive with receptors found in higher concentration on pathognomic cell populations. For example, labeled chemotactic peptides have been investigated for the imaging of focal sites of bacterial infection (2, 13). Oncologic applications of peptides include the use of radiolabeled octreotide analogues for delineation of somatostatin receptor positive tumors (1, 5, 19, 27) and Z31-labeled vasoactive intestinal peptide for the localization of intestinal adenomas and endocrine tumors (35,36). An important feature observed in these studies is the rapid blood clearance of the labeled peptides, making them potentially useful carriers for use with short-lived radionuclides such as 18F.

of peptides and singleOnly a few reports dealing with F-labeling chain antibody fragments have appeared (9, 16, 34). The presence of receptors on melanomas for the tridecapeptide cr-melanocyte stimulating hormone ((r-MSH) has led investigators to explore the possibility of using radiolabeled a-MSH and its analogues as tools for the diagnosis and treatment of this neoplasm (4, 38). Receptors for (Y-MSH have been identified on multiple murine and human melanoma cell lines; however, important differences have been observed between the two species. With murine melanoma cell lines and tissues, dissociation constants in the range of 300 to 1000 pM with 5000 to 20,000 receptors per cell have been reported, whereas most human melanoma lines exhibit an approximately lo-fold lower number of receptors per cell and up to a 5-fold higher affinity (24-26, 28,29). Finally, a-MSH receptors on murine melanoma cell lines undergo rapid internalization, whereas those on human lines do not (24, 25). In the current study, we investigated the feasibility of labeling an (w-MSH analogue with 18F. Because of the inherent instability of a-MSH under physiological conditions, a more stable D-amino acid containing analogue, [Nle4,D-Phe7]-a-MSH, was used (22). The amino acid sequence of this peptide is given in Fig. 1. Recently, we described the labeling of [Nle4,D-Phe7]-cw-MSH with radioiodine using N-succinimidyl 3-[l]iodobenzoate, [1Z51]SlB (15). With respect to in vitro binding properties and in viwo stability, [Nle4,DPhe7,Lys-(251)MIB]-(r-MSH had more favorable characteristics than [NLe4,n-Phe7]-cr-MSH radioiodinated directly on its tyrosine residue (24), as well as (u-MSH labeled either directly or using [r2l]SlB. For this reason, r8F labeling was performed using an analogous reagent, N-succinimidyl4-[Fjfluorobenzoate, [FISFB, which we have used previously for labeling antibody fragments (3 l33) and a chemotactic peptide (34).

Address reprint requests to: Ganesan Vaidyanathan, partment of Radiology, Duke University Medical 27710; E-mail: Accepted 14 October 1996.

PhD, Box 3808, DeCenter, Durham, NC


from Aldrich Chemical Co. analogue [Nle4,D-Phe7]-u-MSH unless was

All chemicals were purchased otherwise noted. The (-r-MSH


G. Vaidyanathan

and M. R. Zalutsky


P = N-Acetyl-Ser-Tyr-Ser-NorLeu-Glu-His-D-Phe-ArgTrp-Gly-Lys-Pro-Val-NH2 ([Nle4,0-Phe7]-a-MSH)
FIG. 1. Reaction scheme for the synthesis of the [Nle4,D-Phe7,Lys-( F)PFB]-wMSH peptide conjugate.

obtained from Sigma Chemical Co. Sodium [251]iodide and sodium [311]iodide with specific activities of 2200 Ci/mmol and 1200 Ci/ mmol, respectively, were obtained from DuPont-New England Nuclear. The [Nle4,D-Phe7]-cy-MSH was labeled with 1 or I using SIB according to the method of Garg et al. (15). Unlabeled SFB was synthesized as reported in a previous publication (32). High-performance liquid chromatography was conducted using one of the following systems: (1) Two LKB Model 2150 pumps, an LKB Model 2152 control system, an LKB Model 2138 fixedwavelength UV-detector, and a Beckman Model 170 radioisotope detector (peak analysis was performed using a Nelson Analytical software package on an IBM computer) and (2) a Perkin-Elmer Series 4 Liquid Chromatograph connected to a Perkin-Elmer LC-95 UV/visible spectrophotometer detector and a Perkin-Elmer LCI100 Laboratory Computing Integrator. For methods programming, a Perkin-Elmer 6312 display terminal was used. Mass spectra were obtained on a JEOL SX-102 high-resolution mass spectrometer. Amino acid analysis and sequencing was performed at the Department of Pathology, Duke University Medical Center Shared Resource Facility. Amino acid analysis was performed using a Beckman 6300 amino acid analyzer. For sequencing, automatic Edman degradation was carried out in an Applied Biosystems 477A sequencer with on-line analysis of the phenylthiohydantoins using an Applied Biosystems 120A HPLC. The murine melanoma cell line B16-Fl was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured at 37C in a humidified atmosphere containing 5% carbon dioxide. The culture medium consisted of modified essential media (MEM) with Earles salts, 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1% MEM nonessential amino acids, 1.5% MEM vitamin solution, 50 units/ml penicillin, and 50 p,g/mL of streptomycin.


of me4,D-Phe]-a-MSH

with SFB

The peptide conjugate [Nle4,D-Phe7,Lys1-PFB]-cr-MSH was prepared for use as an HPLC standard and for the IC,, measurements. To 5 mg (3 kmol) of [Nle4,D-Phe7]-a-MSH in 2 mL of 1% triethylamine/DMF was added 0.7 mg (3 p,mol) of N-succinimidyl 4-fluorobenzoate (SFB) in 100 p,L of the above solvent over a lo-min period. The mixture was stirred at room temperature under argon for 18 to 20 h. Dimethyl formamide was removed by rotary evaporation

using a vacuum pump, and the residue was dissolved in 1 to 2 mL of methanol. The [Nle4,D-Phe7,Lys-PFB]-a-MSH was purified by semi-preparative HPLC using a Waters 25 mm x 100 mm Delta-Pak cartridge column. Eluent A was 0.1% trifluoroacetic acid in water, and eluent B was 0.1% trifluoroacetic acid in 3:l acetonitrile:water. The elution protocol consisted of a gradient running from 30% to 50% B in 15 min followed by an isocratic run at 50% B for another 15 min; the flow rate was 3 mL/min. The ta of [Nle4,D-Phe7,LysPFB]-a-MSH was about 30 min. After evaporation of solvent from the HPLC fractions containing the product, the residue was reconstituted in ethanol. MS (FAB) m/z 1791.4 (M+Na), 1768.4 (M). Amino acid analysis: Arg = 1.01, Glu = 1.00, Gly = 1.11, His = 0.96, Lys = 0.93, Phe = 0.97, Pro = 1.02, Ser = 1.50, Tyr = 1.00, Val = 0.93. The position of attachment of para-fluorobenzoyl moiety was determined by three different experiments. The first was the color development with ninhydrin. Because the a-amino group is protected with an acetyl moiety and none of the side chains other than that of the Lys in [Nle4,D-Phe]-u-MSH will give Rhuemans purple on treatment with ninhydrin, [Nle4,D-Phe,Lys-PFB]-aMSH should not give a positive color test with ninhydrin. Indeed, while [Nle4,D-Phe]-a-MSH gave a dark purple color on treatment with ethanolic ninhydrin, [Nle4,n-Phe7,Lys1-PFB]-a-MSH gave only a faint pink color. Circumstantial evidence for the proposed regiochemistry was obtained from collisional activation link scan FAB mass spectrometry. Subjecting [Nle4,0-Phe7,Lys-PFB]-aMSH to this analysis resulted in one major fragment of 1221 daltons, which corresponds to the loss of -CO-Gly-Lys(4fluorobenzoyl)-Pro-Val-NH,. More definitive proof that the 4-fluorobenzoyl moiety is indeed attached to Lys was obtained by the degradation of the peptide with endoproteinase Glu-C (Protease V8; Boehringer Mannheim) and subsequent sequencing of the major fragment. In ammonium carbonate buffer (pH 7.8), Protease V8 hydrolyzes the peptide specifically at the carboxyl side of glutamate residue. [Nle4,D-Phe, Lys-PFB],cw-MSH was digested with Protease V8 for more than 70 h. A major fragment was isolated by HPLC and subjected to Plasma Desorption Mass Spectrometry. It had a mass (1148 daltons) corresponding to His-Phe-Arg-Trp-Gly-Lys(4fluorobenzoyl)-Pro-Val-NH2. Sequencing of this fragment by Edman degradation identified the presence of His, Phe, Arg, Trp, and Gly. No Lys, Pro, or Val could be identified. Identification of the last couple of amino acid residues is sometimes problematic with








- - q - - Non-soecific


2 Incubation

3 time (Hours)
murine melanoma cells


2. Binding

of [Nle4,D-Phe7,Lys1-(18F)PFB]PcY-MSH

to B16-Fl

as a function

of incubation


this technique. Nonetheless, acylated, the para-fluorobenzoyl sine side chain.

as side chains of Pro and Val cannot be group must be attached to the ly

the product activity was passed through an activated Cl8 solidphase cartridge (Waters tC18). The cartridge was washed with 5 mL of water, and the activity was eluted with 250 p,L portions of ethanol. The majority of activity eluted in fractions 2 and 3. The ethanol from pooled fractions was evaporated and the activity reconstituted in the required buffer.

The [sF]SFB was prepared in three steps beginning with the fluorination of 4-formyl-N,N,N-trimethylanilinium triflate and purified by HPLC as described (33). The HPLC fractions containing 5 to 15 mCi of [18F]SFB were evaporated to a small volume in a rotary evaporator. The activity was transferred to a 1-mL Reacti@ vial and evaporated further with argon in such a way that it was contained in 10 p,L volume at the bottom of the vial. Triethylamine (1 p,L) and a solution of [Nle4,n-Phe7]-a-MSH in DMF (5 (*L of 30-50 mM) were added, vortexed, and left at room temperature for 5 to 10 min. The reaction mixture was injected onto an HPLC system consisting of a Waters I*.-Bondapak Cl8 column (3.9 mm x 300 mm) eluted with a gradient of 30% to 50% of solvent B in 15 min at a flow rate of 1 mL/min (solvent A, 0.1% trifluoroacetic acid in water; solvent B, 0.1% trifluoroacetic acid in 3:l acetonitrile:water). The ta for [Nle4,n-Phe7]-a-MSH and [Nle4,D-Phe,Lys-PFB]a-MSH were 7.5 min and 16.5 min, respectively. The desired peptide conjugate product was isolated in more than 80% decaycorrected yield. After evaporating most of the acetonitrile from the HPLC fractions containing [N]e4,n-Phe7,Lys-(sF)PFB]-a!-MSH,





Three experiments were performed to evaluate the binding characteristics of [Nle4,n-Phe7,Lys-(sF)PFB]-o(-MSH to the a-MSH receptor positive murine melanoma B16-Fl line. All incubation conditions were performed in triplicate. In the first study, the binding of [Nle4,n-Phe7,Lys-(8F)PFB]-a-MSH was studied as a function of time using conditions previously optimized by Siegrist et al. (25). About 250 nCi (-450 Ci/mmol; 0.56 pmol) of [Nle4,n-Phe7,Lys(sF)PFB]-a-MSH was incubated at 15C for 15 min, 1, 2, 3, and 4 h with 2 x lo6 B16-Fl cells in 1 mL of buffer containing 25 mM HEPES, 0.2% bovine serum albumin, and 0.3 mM 1, lophenanthroline in MEM. The nonspecific binding was determined at each time point by co-incubating the cells with 10 FM [Nle4,DPhe7]-a-MSH. After each incubation period, the cells were spun down, washed twice with phosphate-buffered saline, and counted for F activity in an automated gamma counter (LKB 1282; Wallac, Finland). The second experiment compared the binding of


G. Vaidyanathan

and M. R. Zalutsky



Specific Binding Binding

- - 0 - - Non-specific Iodine-l 25

Incubation time (Hours)

FIG. 3. Paired-label binding of [Nle4,0-Pbe,Lys-( murine melanoma cells as a function of incubation lsF)PFB]-cw~MSH time. and [Nle4,D~Pbe7,Lys1-( Z51)MIB]-cxMSH to B16-Fl

[Nle4,D-Phe7,Lys-( F)PFB]-(w-MSH with that of [Nle4,DPhe7,Lys1 -( 1251)MIB]-a,MSH in a paired-label format assay. Cells were incubated as above with about 250 nCi (-1000 Ci/mmol; 0.25 pmol) of [Nle4,0-Phe7,Lys1-(8F)PFB]-~-MSH and 100 nCi (-2000 Ci/mmol; 0.05 pmol) of [Nle4,D-Phe7,Lys11-(251)MIB]-olMSH for 15 min, 1, 2, and 3 h. The cells were counted for 1 and 18F activity using a dual-label program. In parallel, the binding of each radiolabeled [Nle4,D-Phe7]-cr-MSH conjugate was determined in a single-label assay conducted with a 3-h incubation period. Finally, the ICsc values for the displacement of [Nle4,D-Phe7,Lys1 (I)MIB]-a-MSH by [Nle4,D-Phe7,Lys1i-PFB]-cx-MSH and [Nle4,n-Phe7]-a-MSH were determined. In this assay, the binding of [Nle4,D-Phe7,Lys-(311)MIB]-cU-MSH (25 nCi; -1000 Ci/ mmol) to B16-Fl cells was measured in the absence and presence of various concentrations of [Nle4,D-Phe7]-cy-MSH and [Nle4,DPhe7,Lys1-PFB]-o-MSH using a 3-h incubation period.

in normal mice. BALB/c mice were injected via tail vein with 3 pCi (-1.5 pmol) of 251-labeled peptide and 10 pCi (10 pmol) of sFs labeled peptide, and at 1 h and 4 h postinjection, groups of five mice were killed by an overdose of halothane. The animals were dissected and the tissues of interest isolated, washed with saline, blot-dried, standards. The and counted for 1 and 18F along with injection uptake of 1 and F was expressed as the percentage of the injected dose localized per gram of tissue (%ID/g). The statistical significance of the difference in uptake between the two radionuelides was determined using a paired Students t-test. The difference was considered to be significant if p values were less than 0.05.





in Normal

-( sF)PFB]was performed

A paired-label tissue distribution of [Nle4,n-Phe7,Lys1 (Y-MSH and [Nle4,n-Phe7,Lys1 -( 1251)MIB]-~-MSH

The presence of specific receptors on the tumor cell surface has motivated investigators to develop peptide ligands and MAbs reactive with these receptors as radiopharmaceuticals for the detection and possibly the treatment of a variety of cancers. Peptides are particularly attractive for use with 18F because their rapid clearance from normal tissues is more compatible with the physical half-life of this radionuclide. The feasibility of labeling peptides with F had








i-O[Nle4,D-Phe7]-a-MSH i - -0 - - [Nle4,D-Phe7,Lys-PFB]-a-MSH 75



0 -12
-11 -10






FIG. 4. Log-dose response curves the binding of [Nle4, D-Phe7,Lys-( of [Nle4,D-Phe7]McY-MSH 311)MIB]-ar-MSH and to B16eFl

ligand (Log [MI)

[Nle4,0-Phe7,Lys1JTB]-cx-MSH murine melanoma in a competition cells. assay on

been demonstrated both by our group and by others (16, 34). A number of methods are available for labeling peptides and proteins with F (9, 16, 21); however, the results of a recent study suggest that the reagent used in the current investigation, [sF]SFB, may be the method of choice with respect to maximizing conjugation yields and in viwo stability (37). The current study was performed to investigate the feasibility of labeling a tumor-associated receptor-avid peptide with sF. Owing to its rapid degradation under physiological conditions, a-MSH itself is not suitable for in wivo applications. For this reason, this study utilized [Nle4,D-Phe7]-a-MSH, an analogue in which the methionine at position-4 and L-phenylalanine at position-7 of CX-MSH were replaced by norleucine and D-phenylalanine, respectively, substitutions that provide a peptide with higher affinity, lower nonspecific binding, and greater resistance to proteolytic degradation (22, 25). The availability of a wealth of information concerning the interaction of (-w-MSH and its analogues with melanoma cells prompted us to use one of these analogues for the current study ( 11). Although other melanocortin receptors have been identified on normal tissues, the fact that the human a-MSH receptor appears to be expressed only on melanomas and not on normal tissues (8) makes these peptides attractive for the specific targeting of melanomas. Thus, combining the potential tumor specificity of an

a-MSH analogue with PET imaging could provide a powerful tool in the diagnosis of melanomas. However, it is critical to demonstrate that radiofluorination can be accomplished without compromising high-affinity binding to the a-MSH receptor and that the 18F-labeled peptide possesses adequate stability and rapidity of clearance from normal tissues. Derivatization of [Nle4,D-Phe7]-cY-MSH with SFB at the macro level (Fig. 1) required a longer reaction time than that reported earlier for the chemotactic peptide formyl-norleucyl-leucylphenylanalyl-norleucyl-tyrosyl-lysine (34). In the latter case, large excess of SFB could be used because only one amino acid residue reacted with SFB, and a reaction period of 4 to 5 h was required. However, when the same conditions were applied for [Nle4,D-Phe7](Y-MSH, the mass spectrum of the product reflected the presence of two SFB units (data not shown) indicating that, in addition to the lysine, a second amino acid residue had been modified. Gradual addition of one equivalent of SFB overcame this problem, but these conditions necessitated an 18- to 20-h reaction. The results of ninhydrin color test, collisional activation link scan FAB mass spectrometry, and enzymatic degradation/sequencing suggest that the 4-fluorobenzoyl moiety is indeed attached to Lys. With the analogous peptide conjugate, [Nle4,n-Phe7,Lys-(251)MIB]-(Y-MSH, derivatization also occurred at Lys as shown by the fact that iodoben-


G. Vaidyanathan

and M. R. Zalutsky

TABLE 1. Paired-Label Tissue Distribution ing Injection of [Nle4,D-Phe7,Lys1-(8F)PFB]-oL-MSH ( 251)MIB]-~-MSH Percent 1 hr Tissue Liver Spleen Lungs Heart Kidney Bladder Stomach Small intestine Large intestine Thyroid Muscle Bone Blood Brain 2.36 0.38 0.74 0.32 3.18 118.96 1.52 5.46 0.17 0.47 0.46 0.26 0.59 0.07 125 I * f * f k f k k f f f f f f 0.68 0.08 0.22 0.06 0.54 83.55 1.20 0.87 0.04 0.28 0.31 0.10 0.12 0.05 0.80 0.25 0.56 0.17 3.46 164.81 0.74 2.40 0.14 0.42 0.60 0.35 0.32 0.05 sF k f k * k 2 k k k f f f. f f Injected

of lz5 I and


in Normal Mice and [Nle4,D-Phe7,Lys-





Tissue 4 hr


I 0.04 0.04 0.04 0.02 0.03 40.48 0.11 0.05 0.10 0.03 0.04 0.06 0.03 0.04 0.05 0.04 0.12 0.13 0.15 70.95 0.21 0.13 0.22 0.03 0.07 0.14 0.01 0.02

18 F c f f f f. + * * * + f f + + 0.03 0.02 0.16 0.23 0.03b 64.98 0.14 0.04 0.03 O.Olh 0.05ba 0.09 0.00 0.04

0.38 0.07 0.11 0.05 0.67 119.56 0.64 0.42 0.05~bJ 0.39 0.41 0.15b.c 0.12 0.03

0.29 0.13 0.19 0.08 0.34 60.58 0.26 0.20 0.42 0.14 0.07 0.11 0.14 0.04

f f + + f f k + k * + + * k

a Mean + SD (n = 5). bn=4. Difference between the uptake

of the two nuclides


not significant

(p > 0.05).

zoic acid-lysine conjugate was the primary labeled catabolite observed in viva (15). Radiolabeling of [Nle4,D-Phe7]-cy-MSH by reaction with [18F]SFB was straightforward. Unlike our previous experience with the chemotactic peptide formyl-norleucyl-leucyl-phenylanalyl-norleucyl-tyrosyl-lysine (34), solubility of [Nle4,D-Phe7]-cx-MSH in DMF was not a problem, and it was not necessary to use lithium chloride to increase peptide solubility. Radiochemical yields of more than 80% for the synthesis of [Nle4,D-Phe7,Lys-( 8F)PFB]-(r-MSH were obtained routinely. These coupling efficiencies are considerably higher than those observed for [Nle4,D-Phe7,Lys1-( *I)MIB]-a-MSH (15); however, it should be noted that the radioiodination reactions were run at a considerably lower peptide concentration, an important variable governing the efficiency of these acylation agents (39). Because of the relatively low number of a-MSH receptors on melanoma cells (25), maximizing the specific activity of the [Nle4,D-Phe7,Lys1-( F)PFB]-a-MSH peptide conjugate could be important. The specific activity of the [Nle4,D-Phe7,Lys( 18F)PFB]-a-MSH preparations was determined by HPLC comparisons with [Nle4,D-Phe7,Lys1 -PFB]-a-MSH standards. Using a maximum of 80 mCi of [Flfluoride, specific activities ranging from 400 to 1000 Ci/mmol were obtained. The specific activity of [18F]SFB itself was not determined in these studies. Originally, a specific activity of 300 Ci/mmol was reported for [FISFB (32). With our current procedure (33), a substantial reduction in total labeling time has been achieved, and as a result of this and probably other factors (see below), the specific activities of [18F]SFB used in the current studies were presumably higher. The specific activities of the [Nle4,D-Phe7,Lys1-( 18F)PFB]-~-MSH, while similar to that of a F-labeled octreotide (1100 Ci/mmol; 16), were somewhat higher than those reported previously for [sF]SFB labeling of a chemotactic peptide (34) and the labeling of an antibody Fv fragment with Nsuccinimidyl4-( [F]fluoromethyl)benzoate (9). However, it should be noted that considerably higher specific activities for 18F-labeled compounds such as 6-[i8F]fluorodopamine (ZOOO5000 Ci/mmol) have been reported (10). A number of factors may

contribute to the varying specific activities observed. The amount of carrier fluoride has been found to increase with repeated use of [sO]HzO (23). Another source of carrier fluoride is the substrates containing fluorines in their structure (17). The number of steps involved in the chemistry, or more importantly the duration of labeling, will affect the specific activity of the final labeled agent. Obviously, the specific activity can also vary with varying amounts [F]fluoride activity used. The in vitro binding characteristics of [Nle4,0-Phe7,Lys( 18F)PFB]-a-MSH to B16-Fl murine melanoma cells appeared to be influenced by the specific activity of the labeled preparations. The first assay, performed with 450 Ci/mmol 18F-labeled peptide, was done to determine the kinetics of binding to an (w-MSH receptorpositive cell line. As shown in Fig. 2, the percent of activity bound to B16-Fl cells increased with time and plateaued at about 2 h. This behavior was similar to that reported previously for the binding of a radioiodinated a*MSH analogue to this cell line (24). However, the maximum specific binding of [Nle4,D-Phe7,Lys -( F)PFB]-cxMSH was only about 34%, a value about 10 times lower than that obtained with [Nle4,D-Phe7,Lys11-(251)MIB]-(Y-MSH when the assay was performed under identical conditions (15). Although the absolute level of [Nle4,D-Phe7,Lys-( 18F)PFB]-aMSH binding was low, the specific/nonspecific binding ratio was relatively high, about 2O:l. This behavior was not unexpected; similar results were obtained with [Nle4,D-Phe7,Lys-(251)MIB]-olMSH (15). This is an important characteristic, for many r~-MSH analogues radioiodinated at Tyr7, particularly those with a norleutine-for-methionine substitution at position 4, have nonspecific binding percentages of 20% or more (12, 15). The low level of absolute binding of [Nle4,D-Phe7,Lys(*F)PFB]-a-MSH could be due to either alteration in receptor binding related to structural modification of the peptide or to saturation of cr-MSH receptors by an adventitious carrier present in the preparation. The latter explanation seems more probable given the low apparent K,, measured for the analogous SIB peptide conjugate (15). In addition, if 10,000 a-MSH receptors per 816sFl cell are







assumed (25), then under these assay conditions, the B,,, was sites. At a specific activity of 450 equivalent to 2 x 10 binding Ci/mmol, 250 nCi of i8F-labeled peptide would correspond to about 3.3 x 10 peptide molecules, suggesting that the maximum percentage of [Nle4,n-Phe7,Lys-( F)PFB]-a-MSH that could have bound would have been about 6%. The results of a second assay performed with [Nle4,D-Phe7,Lys(18F)PFB]-cr-MSH (-1000 Ci/mmol; 0.25 pmol) and [Nle4,DPhe7,Lys1-(251)MIB]-a-MSH (-2000 Ci/mmol; 0.05 pmol), incubated together and separately, support the belief that specific activity is an important factor influencing the in vitro binding of the F-labeled peptide. When the peptides were incubated separately for 3 h, the specific binding percentages were 11 f 2% and 25 f 5%, respectively. As expected, the twofold increase in specific activity of [Nle4,n-Phe7,Lys11-(8F)PFB]-a-MSH from the first to the second experiment resulted in a significant increase in specific binding; however, the level of binding achieved was still less than that of the 251-labeled (w-MSH analogue. Results of the paired-label binding assay are shown in Fig. 3. Interestingly, under these conditions the maximum specific binding of [Nle4,D-Phe7,Lys-( 18F)PFB]-~-MSH at 3 h was 9 + 2%, a level significantly higher than that observed for [Nle4,D-Phe7,Lys-(251)MIB]-a-MSH, 5 + 2% (p < 0.05). The higher binding of the 18F-labeled peptide under conditions of competitive binding suggests that [Nle4,D-Phe7,Lys1-PFB]-(-Y-MSH has a higher affinity for a-MSH receptors, a characteristic consistent with the smaller size of the halobenzoyl group conjugated to [Nle4,n-Phe7]-a-MSH. A final binding assay was performed to compare the IC,, values for the competitive inhibition of [Nle4,D-Phe7,Lys1-(311)MIB]-aMSH binding to B16-Fl melanoma cells by [Nle4,D-Phe7,LysPFB]-(w-MSH and unmodified [Nle4,D-Phe7]-a-MSH (Fig. 4). The IC,, value for [Nle4,0-Phe7,Lys-PFB]-(-w-MSH, 112 f 22 pM, was not significantly different from that of [Nle4,D-Phe7]-o-MSH (89 ? 9 PM) suggesting that conjugation of para-fluorobenzoate to [Nle4,n-Phe7]-a-MSH did not compromise the affinity of the peptide. The IC,, value obtained for [Nle4,n-Phe7]-a-MSH was higher than that reported earlier (15), a difference that probably reflects variation in assay conditions between the two experiments. A likely factor is that the [Nle4,D-Phe7,Lys1-(1311)MIB]-o-MSH preparation used in the current study had a specific activity about twofold lower than the [Nle4,D-Phe7,Lys-( 1251)MIB]-u-MSH used previously. When the competitive inhibition data were replotted as Hill curves, Hill coefficients of less than 1 were obtained. This suggests negative cooperativity or that the radioligand is binding to multiple sites (30). For in viwo applications, a potential advantage of labeling peptides compared with MAbs is their rapid clearance from normal tissues. However, this behavior is often dependent on the radionuclide and the labeling method used. For example, retention of activity in liver and kidneys has been observed with several In-labeled CX-MSH analogues (3, 4). A paired-label experiment was performed in normal mice to compare the tissue distribution of [Nle4,D-Phe7,Lys1(F)PFB]-(u-MSH and [Nle4,0-Phe7,Lys1-(1251)MIB]-a-MSH. As summarized in Table 1, the normal tissue clearance was rapid for both radionuclides. Although metabolic cages were not used to permit total urine collection, a high activity concentration of both tracers was found in urine obtained from the mice at necropsy. At 1 h postinjection, the tissue with the highest activity was the kidney (18F, 3.46 f 0.67% ID/g; *I, 3.18 f 0.54% ID/g, difference not statistically significant). Relatively high uptake was also observed in the small intestine; however, in this case, the uptake of 18F (2.40 + 0.42% ID/g) was more than twofold less (p < 0.05) than that of 25I

(5.46 + 0.87% ID/g). Liver uptake of F also was significantly less than that of 1251. Bone uptake after injection of [Nle4,D-Phe7,Lys1(18F)PFB]-a-MSH declined from 0.35 f 0.15% ID/g at 1 h to 0.14 + 0.09% ID/g at 4 h, suggesting that defluorination of the peptide and/or its labeled catabolites was minimal. The uptake of [Nle4,0-Phe7,Lys-( 18F)PFB]-a-MSH in various tissues at 1 h was qualitatively similar to that seen in the case of F-labeled chemotactic peptide (34). In addition, normal tissue activity levels were much lower than those reported for several In-labeled and radioiodinated u-MSH analogues (3, 4, 15). It seems likely that the generally lower tissue uptake of [Nle4,DPhe7,Lys -( 18F)PFB]-(u-MSH in comparison with [Nle4,nPhe7,Lys1-(251)MIB]-(r-MSH may be due to the lower lipophilicity of the former and its labeled catabolites. Under identical reversephase HPLC conditions, [Nle4,D-Phe7,Lys1-MIB]-tx-MSH had a higher ta than [Nle4,D-Phe7,Lys-PFB]-o-MSH (data not shown). However, it should be pointed out that, while the kidney uptake of 18F and 1 was not significantly different at lh, at 4h kidney uptake of 1251 was more than twice (I, < 0.05) that of 18F. In summary, [Nle4,D-Phe7]-a-MSH could be labeled with F in excellent radiochemical yields using [18F]SFB. Derivatization of this a-MSH analogue with SFB had minimal effect on its affinity for (w-MSH receptors on melanoma cells. The rapid rate of 18F activity clearance of [Nle4,D-Phe7,Lys-(18F)PFB]-cx-MSH from normal tissues should be compatible with the short half-life of this radionuelide. However, because of the low number of rx-MSH receptors on melanoma cells, it remains to be ascertained whether [Nle4,DPhe7,Lys1-( F)PFB]-c-r-MSH can be prepared with sufficient specific activity to permit the use of this tracer for the PET imaging of melanomas. The authors would like to acknowledge the excellent technical assistance of Donna Afleck, Kevin Alston, Susan Side, and Philip Welsh. This work was supported by Grant DE-FG02-96ER62148 from the U.S. Department of Energy.

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